Most blood analyzer systems in use today count platelets by means of electrically and/or optically based measurements. In an electrical or impedance-based measurement system, particles within a carrier fluid passing through a detection aperture of a flow cytometer cause the generation of electrical pulses proportional to their volume. Using a thresholding technique on the pulses, the particles that gave rise to the pulses are classified as platelets, erythrocytes (red blood cells or RBCs), or leukocytes (white blood cells or WBCs) and debris. Although, in most cases, an electrically based measurement system provides relatively accurate results, it is not without its limitations.
For example, an impedance-based measurement analyzer is not capable of discriminating between platelets and interfering particles, such as microcytes, schistocytes (fragmented RBCs), debris and electronic noise, which leads to falsely high platelet counts. Conversely, platelet aggregates and oversized platelets may fall outside an upper size threshold and be classified as RBCs, which leads to falsely low platelet readings.
In order to determine the course of treatment of a patient, it is generally mandated clinical practice to obtain a very accurate platelet count. For example, if the platelet count is extremely low (e.g., in a range of from 5,000 to 20,000 platelets per microliter), it may become necessary to transfuse platelets or to forego surgery. On the other hand, the critical threshold for a pregnant woman is considerably higher (e.g., favorable platelet counts should be in excess of 140,000 platelets per microliter).
In a typical hematology analyzer, a mathematical fit is performed on the platelet volume distribution between two and twenty fL in order to expand the dynamic range, e.g., up to seventy fL. Except for a platelet count in which part of the distribution is derived, an accurate platelet count cannot be obtained if the raw data does not fit a logarithmic distribution, the mean platelet volume is outside its normal range, the mode is outside its normal range, or if the upper end of the platelet distribution is not decreasing. In such instances, the sample is flagged, and only those platelets lying in the two to twenty femtoliters (fL) range are reported.
Difficulty in obtaining an accurate platelet count at relatively low levels may also be due to poor discrimination of DC noise, smaller RBCs, and coincident passage of RBCs and platelets through the flow cell's detection aperture. Platelet distributions can also shift with respect to volume, depending upon the age of the platelets, thereby changing the shape of the distribution and potentially leading to incorrect extrapolations; since the average lifetime of platelets is only three to five days, the shift can be fairly rapid. As described below, resolution of platelets by other (e.g., optically based) techniques, such as differential light scattering, can help to reduce these discrepancies.
Examples of various types of optically-based prior art schemes for counting platelets include the system described in the U.S. patent to Bessis et al, U.S. Pat. No. 3,955,890, which details a system to collect scattered light and measure its intensity at a minimum of two different azimuthal angles. This patent discloses that while spherical particles produce circular scattering patterns, an elongated RBC passing through a detection aperture scatters light in an ellipsoidal pattern, which is oriented in such a way that its major axis is perpendicular to the major axis of the elongated cell. Consequently, this system allows a determination of light scatter ratios for angles collected perpendicular to one another, as well as determining the length-to-width ratios for individual RBCs and other cellular entities.
U.S. Patent to Weiner et al, U.S. Pat. No. 4,202,625 describes a system for using light scatter to discriminate RBCs by amplifying noise, platelet pulses and RBC pulses, and comparing them to a predetermined noise threshold. The relative amplitudes of the respective signals represent either platelets or RBCs.
U.S. Patent to Ornstein et al, U.S. Pat. No. 4,412,004 discloses a method for isovolumetrically sphering and fixing RBCs, thereby eliminating the effect of shape on the determination of their respective and collective volumes, as determined by using different angles of forward light scatter. This technique is also addressed in the publication Cytometry 3/6, 1983, pp 419-427, which describes that, by using high and low angle light scatter, both normal and abnormal RBC samples produce greater separation between sphered RBCs and platelets, as compared to RBCs that are unsphered.
U.S. Patent to Hansen, U.S. Pat. No. 4,577,964 describes a system wherein low angle scatter is used to discriminate platelets from RBCs, based on cell volume, cell refractivity, and duration of the scattered light.
In another patent relating to the determination of leukocytes and nucleated red blood cells (NRBC), U.S. Pat. No. 5,559,037 (to Kim et al.) discloses a method for flow cytometric analysis of nucleated red blood cells and leukocytes. The method comprises lysis of red blood cells and NRBC cytoplasm from a whole blood sample to expose the nucleated red blood cell nuclei to a vital nuclear stain and minimizing the permeation of the vital nuclear stain into the leukocytes and analyzing the sample by measuring fluorescence and two angles of light scatter. This method features a triple triggering method which blocks signals from debris (fluorescent and non-fluorescent) and identifies the signals which fall below the axial light loss (ALL) trigger but above the fluorescence trigger (FL3) as NRBCs. This method requires heating of the reagent to 42° C. in order to obtain the NRBC and leukocyte differentiations.
U.S. Patent to Frank et al, U.S. Pat. No. 5,798,827, describes a system for determining the shape of individual RBCs by measuring the asymmetry of the light scatter pattern produced by laser illumination of the cell collected in specific angular quadrants.
U.S. Patents to Zelmanovic et al, U.S. Pat. Nos. 5,817,519 and 6,025,201 describe light scattering-based platelet discrimination systems, in which light scattering is measured over a high angle interval of five to twenty degrees and a low angle interval of one to five degrees. First and second optical channel light scattering signals are converted to platelet volume values and a refractive index of the platelets, which is converted into a platelet component concentration value. A platelet dry mass value is calculated as a product of the platelet component concentration value and the platelet volume. Histograms are then formed of the platelet volumes, the platelet component concentrations and the platelet dry masses. Platelets are resolved from non-platelets, and platelet parameters are determined by the presence of light scatter-based platelet signals within a volume versus refractive index map.
U.S. Patent to Gill et al, U.S. Pat. No. 5,891,734 describes an automated hematology and fluorescent cytometry system that detects and counts RBCs and platelets using an impedance flow transducer, and also uses multi-angle light scatter and fluorescence to count and differentiate platelets or platelet aggregates or both within an optical flow transducer.
U.S. Patent to Horton et al, U.S. Pat. No. 6,060,322 details an optical system for identifying reticulated RBCs and platelets through the use of a reagent composition that contains a nucleic acid dye and a sphering agent.
U.S. Patent to Kubota, U.S. Pat. No. 6,133,995 describes a light scattering-based blood analyzer, which generates a distribution diagram on the basis of a plurality of parameters extracted from each particle in a sample. A cluster including platelets is separated from other information in the distribution diagram, and a discriminator calculates a discrimination function for the separated cluster including the platelets, and for discriminating the platelets from other particles in the cluster on the basis of the distance from the calculated discrimination function, to produce a count of the number of platelets.
From the above-listed patents and from commercially available equipment for conducting optical platelet measurements, it will be appreciated that optically-based platelet measurement systems employ several approaches for performing optical platelet counts. Typically, optical-based schemes employ at least one low angle forward scatter measurement to determine the volume of the particle.
It should be noted that when light interacts with a particle, some of the incident light changes direction through the scattering process (i.e. light scatter) and part of the light is absorbed by the particles. Both of these processes remove energy from the incident beam. When viewed along the incident axis of the beam, the light loss is called extinction or axial light loss.
The above-referenced patent to Weiner et al teaches that red cells and platelets can be separated using light scatter, and it should be noted that light scatter and light loss are physically different measurements. This is no different than the distinction regarding fluorescence signals, which is another resultant signal produced when light interacts with a particle. For further edification attention may be directed to the publication by H. C. van de Hulst, “Light Scattering by Small Particles”, copyright 1957, Dover Publications Inc., NY.
As a non-limiting example, the Ortho ELT-8 system uses a single light scatter measurement. The CELL-DYN® 4000 manufactured by Abbott Laboratories, the assignee of the above-referenced Gill et al Patent, couples a second scatter measurement, orthogonal light scatter (SS), to determine the internal complexity of the particle in an effort to eliminate non-platelet particles from the platelet count. The Advia 120 system, manufactured by Bayer Corp., the assignee of the above-referenced Patents to Zelmanovic et al, adds a second forward light scatter measurement, which determines the particle's index of refraction for platelet identification. The XE-2100 system manufactured by Sysmex Corp., the assignee of the above-referenced Kubota Patent, identifies platelets through an additional fluorescent measurement of cells stained with a fluorescent dye. It should be noted that it is generally recognized that immunological identification of platelets allows counting of true platelets in the presence of large numbers of confounding particles. The above-referenced CELL-DYN 4000 also allows for an immunological measurement using monoclonal antibody CD-61.